Free fatty acids (FFAs) are energy-rich molecules capable of serving as precursors for the production of liquid transportation fuels and high-value oleochemicals. Fuel properties are dictated by the aliphatic chain length and degree of saturation of the FFA precursors. Medium-chain (C6-C12) FFA feedstocks can be converted to hydrocarbons with fuel properties comparable to gasoline, diesel, or jet fuel. See, for example, Choi Y J & Lee S Y (2013) “Microbial production of short-chain alkanes,” Nature 502(7472):571-574; and Lee S K, Chou H, Ham T S, Lee T S, & Keasling J D (2008) “Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels,” Curr Opin Biotech 19(6):556-563. Fuels derived from microbially produced FFAs would facilitate reduction of the carbon footprint and, unlike bioethanol, avoid expensive and laborious infrastructure and engine remodeling. (Howard T P, et al. (2013) “Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli,” PNAS 110(19):7636-7641.
Escherichia coli is a popular microbial host for FFA production because of its established type II fatty acid biosynthesis (FAB) pathway, short doubling time, and genetic tractability. The E. coli FAB pathway is initiated by the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. Subsequently, CoA is exchanged with acyl carrier protein (ACP), the recognition tag of FAB, producing malonyl-ACP. Malonyl-ACP and acetyl-CoA are condensed to yield acetoacetyl-ACP. The alkyl chain of the β-ketoacyl-ACP is successively extended by two carbon atoms that originate from additional malonyl-ACP. This cycle is terminated by the acyl-ACP thioesterase, which hydrolyzes the thioester bond to generate the FFA and ACP. The specificity of the acyl-ACP thioesterase controls the terminal aliphatic chain length and chemical properties of the FFA product composition. Regulation of the FFA chain length produced through the FAB pathway has typically been achieved by the overexpression of the two native E. coli thioesterases (TesA and TesB), or heterologous expression of various plant and bacterial thioesterases (see Table 1, below), which exhibit a wide range of substrate specificities See Choi & Lee (2013), supra, as well as Steen E J, et al. (2010) “Microbial production of fatty-acid-derived fuels and chemicals from plant biomass,” Nature 463(7280):559-U182; Jing F Y, et al. (2011) “Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity,” BMC Biochem 12:44; Zhang, Li, Agrawal, & San (2011) “Efficient free fatty acid production in Escherichia coli using plant acyl-ACP thioesterases,” Metabolic Engineering 13(6):713-722; Lu, Vora & Khosla (2008) “Overproduction of free fatty acids in E. coli: implications for biodiesel production,” Metabolic Engineering 10(6):333-339; Voelker T A & Davies H M (1994) “Alteration of the specificity and regulation of fatty acid synthesis of Escherichia coli by expression of a plant medium-chain acyl-acyl carrier protein thioesterase,” J Bacteriol 176(23):7320-7327; and Dormann, Voelker, & Ohlrogge (1995) “Cloning and Expression in Escherichia coli of a Novel Thioesterase from Arabidopsis-Thaliana Specific for Long-Chain Acyl-Acyl Carrier Proteins,” Arch Biochem Biophys 316(1):612-618.
Several of these thioesterases have been evolved to further diversify the gamut of attainable FFA compositions. Despite this diversification, very few thioesterases are specific towards a unique aliphatic chain length. Of these studied thioesterases, TesA (a cytosolic TesA that lacks the N-terminal signal peptide and whose crystal structure has been elucidated) produces one of the highest FFA titers. See Steen (2010) and Choi & Lee (2013), supra, and Cho & Cronan (1993) “Escherichia coli Thioesterase-I, Molecular-Cloning and Sequencing of the Structural Gene and Identification as a Periplasmic Enzyme,” Journal of Biological Chemistry 268(13):9238-9245 and Lo, Lin, Shaw, & Liaw (2005) “Substrate Specificities of Escherichia coli Thioesterase I/Protease I/Lysophospholipase L1 Are Governed by Its Switch Loop Movement,” Biochemistry 44(6):1971-1979. In spite of these clear advantages, 'TesA has broad substrate specificity that necessitates costly downstream separation (Steen (2010) and Choi & Lee (2013), supra).
Acyl-acyl carrier protein (Acyl-ACP) thioesterases play an essential role in chain termination during de novo fatty acid synthesis. These thioesterases terminate fatty acyl group extension by catalyzing the hydrolysis of an acyl group on a fatty acid. Thus, because acyl-ACP thioesterases catalyze termination of the iterative chain extension process, the action of acyl-ACP thioesterases determines, in large part, the ultimate carbon chain length of the fatty acids found in any given wild-type organism. See, for example, Aubrey Jones, H. Maelor Davies, and Toni A. Voelker (1995) “Palmitoyl-Acyl Carrier Protein (ACP) “Thioesterase and the Evolutionary Origin of Plant Acyl-ACP Thioesterases,” The Plant Cell, 7:359-371.
The carbon chain length of fatty acids is economically significant because the natural occurrence of certain types of fatty acids, such as medium-chain fatty acids (carbon chain of 6 to 12 carbon atoms) in general and C8 carbon chain length fatty acids in particular, is notably less than long-chain fatty acids (carbon chain longer than 12 carbon atoms). C8 fatty acids are also notable because they are both renewable and also suitable as a precursor to liquid transportation fuels, i.e., biofuel.
Biofuels such as biodiesel are biodegradable, clean-burning combustible fuels made of medium- to long-chain alkanes and esters. Biodiesel can be used in most internal combustion diesel engines in either a pure form, which is referred to as “neat” biodiesel, or as a mix in any concentration with regular, petroleum-derived diesel. An advantage of biodiesel is that it can be generated from renewable, non-petroleum sources. Current methods of making biodiesel involve transesterification of triacylglycerides (mainly vegetable oil). However, this leads to a product comprising a mixture of fatty acid esters and glycerin as an unwanted by-product. In short, because transesterification yields heterogeneous product and an unwanted glycerin by-product, transesterification encompasses unavoidable economic inefficiencies. In addition, the presence of methyl esters and ethyl esters in traditional biodiesel leads to unwanted gelation properties at temperature below about 0° C.
PCT Publication No. WO 2007/136762, published Nov. 29, 2007, to Keasling et al., discloses recombinant microorganisms that are capable of synthesizing products derived from the fatty acid synthetic pathway, including fatty acid esters and fatty alcohols.
PCT Publication No. WO 2008/119082, published Oct. 2, 2008, to Hu et al., discloses genetically engineered cells and microorganisms that produce products from the fatty acid biosynthetic pathway. The products are noted as being particularly useful as biofuels. The Hu et al. publication describes recombinant cells that utilize overexpression of acyl-CoA synthetase enzymes to more efficiently produce fatty acid derivatives.
U.S. Pat. No. 5,955,329, issued Sep. 21, 1999, to Yuan et al., discloses genetically engineered plant acyl-ACP thioesterase proteins having altered substrate specificity. The engineered acyl-ACP thioesterase exhibited an altered substrate specificity as compared to the wild-type acyl-ACP thioesterase.
U.S. Pat. No. 8,617,856, issued Dec. 31, 2013, to Pfleger and Lennen, describes transformed hosts for overproducing fatty acids. The hosts include an exogenous nucleic acid encoding a thioesterase and, optionally, an exogenous nucleic acid encoding an acetyl-CoA carboxylase, wherein an acyl-CoA synthetase in the host is functionally deleted. The hosts preferably include the nucleic acid encoding the thioesterase at an intermediate copy number.
U.S. Pat. No. 9,175,234, issued Nov. 3, 2015 to Hom et al. describes an engineered thioesterase enzyme which converts a C10, C12, or C14 acyl-ACP substrate to a fatty acid derivative with a greater activity as compared to a wild-type thioesterase enzyme. This particular mutant thioesterase has a substitution at an amino acid position selected from the group consisting of positions 78, 80, 101, 108, 111, 117, 118, 122, 145, 152, and 178.
SEQUENCE LISTSEQ ID NO: 1 (amino acid sequence of mature E. coli ‘TesA; PDB 1U8U):1adtllilgds lsagyrmsas aawpallndk wqsktsvvna sisgdtsqqg larlpallkq61hqprwvlvel ggndglrgfq pqqtegtlrq ilqdvkaana epllmqirlp anygrrynea121fsaiypklak efdvpllpff meevylkpqw mqddgihpnr daqpfiadwm akqlqplvnh181dslehhhhhh* *NB: The C-terminal eight (8) residues in SEQ ID NO: 1 is an expression tag. SEQ ID NO: 2: (nucleotide coding sequence, E. coli ‘TesA gene):1atgatgaact tcaacaatgt tttccgctgg catttgccct tcctgtttct ggtcctgtta61accttccgtg ccgccgcagc ggacacgtta ttgattctgg gtgatagcct gagcgccggg121tatcgaatgt ctgccagcgc ggcctggcct gccttgttga atgataagtg gcagagtaaa181acgtcggtag tcaatgccag catcagcggc gacacctcgc aacaagggct ggcgcgcctt241ccggctctgc tgaaacagca tcagccgcgt tgggtgctgg ttgaactggg cggcaatgac301ggtttgcgtg gttttcagcc acagcaaacc gagcaaacgc tgcgccagat tttgcaggat361gtcaaagccg ccaacgctga accattgtta atgcaaatac gtctgcctgc aaactatggt421cgccgttata atgaagcctt tagcgccatt taccccaaac tcgccaaaga gtttgatgtt481ccgctgctgc ccttttttat ggaagaggtc tacctcaagc cacaatggat gcaggatgac541ggtattcatc ccaaccgcga cgcccagccg tttattgccg actggatggc gaagcagttg601cagcctttag taaatcatga ctcataa Nucleotides 79 to the end of SEQ ID NO: 2 (nt 624) encodes the mature, wild-type‘TesA peptide shown in SEQ ID NO: 1.